Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels

ABSTRACT

Devices and methods for detecting an analyte are provided. Devices for voltage sensing of analytes may comprise a fluidic channel defined in a substrate, a pair of sensing electrodes disposed in a fluidic channel for sensing voltage therein, and a pair of electromotive electrodes for applying potential along the fluidic channel. The pair of sensing electrodes may include a first and second sensing electrode disposed at two discrete locations along the length of the fluidic channel and the pair of electromotive electrodes may be disposed at a first end and a second end of the fluidic channel. The fluidic channel may include a nanochannel or a microchannel. Methods for detecting an analyte may include the steps of disposing the analyte in a fluidic channel; applying a potential along the fluidic channel to generate an electrophoretic force therein such that the analyte is translocated from a first end of the fluidic channel to a second end of the fluidic channel; and measuring a voltage signal between a pair of sensing electrodes disposed in the fluidic channel as the analyte moves past the sensing electrodes.

RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Ser. No. 61/093,885, filed Sep. 3, 2008; theentirety of which is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates generally to biopolymer sequencing. Moreparticularly, in certain embodiments, the invention relates todetermining the length of biopolymers and the distances of probes boundto the biopolymer.

BACKGROUND

The technique known as Coulter counting was first proposed by Wallace H.Coulter in the late 1940s as a technique for the high speed counting ofred blood cells. Also referred to as resistive pulse sensing, Coultercounting may be used to measure physical parameters of analytes inelectrolyte solution including size (volume), charge, electrophoreticmobility and concentration. In this technique, two reservoirs ofsolution are separated by a fluidic constriction of known dimensions.The application of a constant DC voltage between the two reservoirsresults in a baseline ionic current that is measured. The magnitude ofthe baseline current is related to the conductivity of the electrolyte,the applied potential, the length of the channel, and thecross-sectional area of the channel. If an analyte is introduced into areservoir, it may pass through the fluidic channel and reduce theobserved current due to a difference in conductivity between theelectrolyte solution and analyte. The magnitude of the reduction incurrent depends on the volume of electrolyte displaced by the analytewhile it is in the fluidic channel.

A benefit of the resistive pulse sensing technique is that it may bescaled down to enable the detection of nanoscale analytes through theuse of nanoscale fluidic constrictions. This capability led to thedevelopment of solid-state nanopores for detecting nanoscale moleculessuch as DNA.

In the case of DNA translocation through a nanopore, the physicaltranslocation is driven by the electrophoretic force generated by theapplied DC voltage. This driving force and the detected signal are,therefore, typically inseparably coupled. The decoupling of these twoeffects may be desirable because the optimal potential for physicaltranslocation is different from that of optimal measurement.

Transverse electrodes have been proposed to provide a transverseelectric field and electric current to sense biomolecules confined in ananofluidic channel. See Liang and Chou 2008 Liang, X; Chou, S. Y.,Nanogap Detector Inside Nanofluidic Channel for Fast Real-TimeLabel-Free DNA Analysis. Nano Lett. 2008, 8, 1472-1476, which isincorporated herein by reference in its entirety. The analytes are movedthrough the channel with an electrophoretic force generated bycurrent-carrying electrodes at the ends of the nanochannel, thereforedecoupling the measurement from the translocation speed.

SUMMARY

Embodiments of the present invention provide devices and methods thatuse electrodes to sense voltage changes, rather than to generatetransverse electric currents, thereby reducing degradation of theelectrodes. In particular, a device described herein utilizeslongitudinally displaced electrodes for electronic sensing ofbiomolecules and other nanoscale analytes in fluidic channels.Embodiments enable characterization of nanoscale analytes, including,e.g., analysis of DNA strands having probes attached thereto.

More particularly, embodiments of the present invention may utilizesensing electrodes for electronic sensing of analytes, e.g., DNA, influidic channels. The sensing electrodes in the fluidic channel may beused to determine the length of the analyte or they may be used todetermine the distance between probes hybridized to a target strand ofDNA. The device design is similar to nanochannel devices used foroptical detection. Two micro-scale liquid reservoirs may be fabricatedat a distance of 100 to 200 μm. One or more fluidic channels may connectthe two reservoirs. A cap may be fabricated by drilling holes that willallow fluid introduction to each reservoir and to provide access formacroscopic electrodes. In use, a voltmeter may be used to monitor thepotential difference between two sensing electrodes.

The DNA to be analyzed may be introduced to one of the microfluidicreservoirs. Macroscopic electrodes may be connected to a power supplyand used to apply a potential between the two reservoirs. DNA fragmentsmay be electrophoretically driven from the microscopic reservoir intothe nanochannels. As each DNA fragment moves down the fluidic channel,it may enter and exit the pair of sensing electrodes disposed in thefluidic channel.

In the absence of DNA, the fluidic channel contains only the ionicsolution and typically have a baseline potential difference measuredbetween the two sensing electrodes. As DNA enters the fluidic channel,the potential measured between the two sensing electrodes may changebecause the DNA has a conductivity different from that of the ionicsolution. When DNA enters the fluidic channel, the conductivity of thechannel between the two sensing electrodes will typically be reduced asDNA is less conductive than the buffer solution (See de Pablo, P. J.;Moreno-Herrero, F; Colchero, J.; Gomez-Herrero, J.; Herrero, P.; Baro,A. M.; Ordejon, P.; Soler, J. M.; Artacho, E. Absence of dc-Conductivityin Phys. Rev. Lett. 2000, 85, 4992-4995, which is incorporated byreference in its entirety). When a portion of the DNA that has a probehybridized to the DNA enters the fluidic channel the potential maychange further. The measured signal may be analyzed to determine thelength of the DNA and/or distances between probes.

In an aspect, an embodiment of the invention includes a device forvoltage sensing of analytes. The device may include a fluidic channeldefined in a substrate, and a pair of sensing electrodes disposed in thefluidic channel for sensing voltage therein. The pair of sensingelectrodes may include a first and a second sensing electrode disposedat two discrete locations along a length of the fluidic channel. A pairof electromotive electrodes may be disposed at a first end and a secondend of the fluidic channel for applying a potential along the fluidicchannel. The fluidic channel may include or consist essentially of ananochannel or a microchannel.

One or more of the following features may be included. The substrate mayinclude or consist essentially of silicon, silicon dioxide, fusedsilica, and/or gallium arsenide. Each of the sensing and electromotivepairs of electrodes may include or consist essentially of platinum,gold, chrome, titanium, silver chloride, silver, and graphene.

The first sensing electrode may be disposed on a first side of thefluidic channel and the second sensing electrode may be disposed on anopposing side of the fluidic channel. Each of the first and secondsensing electrodes may be disposed on a first side of the fluidicchannel, or each of the first and second sensing electrodes maytransverse the fluidic channel. The first sensing electrode maytransverse the fluidic channel and the second sensing electrode may bedisposed on a side of the fluidic channel.

The pair of electromotive electrodes may include macroscopic electrodesfor generating a constant, changing, or oscillating electrophoreticforce in the fluidic channel for translocation of an analyte disposedtherein.

A measurement tool, such as a voltmeter, may be provided for measuring avoltage sensed by the pair of sensing electrodes. The device may includea plurality of fluidic channels. A voltage amplifier may be disposed onthe substrate.

The fluidic channel may have a width selected from a range of 1 nm to 5μm, a depth selected from a range of 1 nm to 5 μm, and/or a lengthselected from a range of 1 μm to 10 cm.

The description of elements of the embodiments of other aspects of theinvention may be applied to this aspect of the invention as well.

In another aspect, the invention features a method for detecting ananalyte, the method including disposing the analyte in a fluidicchannel. A potential is applied along the fluidic channel and theanalyte is translocated from a first end of the fluidic channel to asecond end of the fluidic channel. A voltage signal is measured betweena pair of sensing electrodes disposed in the fluidic channel as theanalyte moves past the pair of sensing electrodes, with the pair ofsensing electrodes including a first and a second electrode disposed attwo discrete locations along a length of the fluidic channel. Thefluidic channel may include or consist essentially of a nanochannel or amicrochannel.

The potential applied along the fluidic channel may include generatingan electrophoretic force therein. Translocating the analyte may includeusing a pressure differential and/or a chemical gradient.

One or more of the following features may be included. The analyte mayinclude a biopolymer, such as a deoxyribonucleic acid, a ribonucleicacid, and/or a polypeptide. The biopolymer may include or consistessentially of a single-stranded molecule. The analyte may include orconsist essentially of a biopolymer having at least one probe attachedthereto.

The voltage signal may change when the biopolymer moves through a volumebetween the sensing electrodes and further change when the portion ofthe biopolymer containing the probe moves through the volume between thesensing electrodes. A time between voltage signal changes may berecorded. A duration of a change in the voltage signal may indicate apresence of a probe, and the voltage signal may be used to determine adistance between two probes on the biopolymer.

A duration of a change in the voltage signal may be used to determine alength of the analyte. Multiple pairs of sensing electrodes may be usedto measure a single analyte molecule as it passes through the fluidicchannel.

The description of elements of the embodiments of other aspects of theinvention may be applied to this aspect of the invention as well.

In yet another aspect, a method for determining a sequence of abiopolymer may include preparing an analyte including the biopolymer.The analyte may be disposed in a fluidic channel. A potential may beapplied along the fluidic channel and the analyte is translocated from afirst end of the fluidic channel to a second end of the fluidic channel.A voltage signal may be measured between a pair of sensing electrodesdisposed in the fluidic channel as the analyte moves past the pair ofsensing electrodes, the voltage signal corresponding to locations alongthe biopolymer, the pair of sensing electrodes including a first and asecond electrode disposed at two discrete locations along a length ofthe fluidic channel. The fluidic channel may include or consistessentially of a nanochannel or a microchannel.

The potential applied along the fluidic channel may include generatingan electrophoretic force therein. The analyte may be translocated byusing a pressure differential and/or a chemical gradient.

One or more of the following features may be included. Preparing theanalyte may include hybridizing the biopolymer with a probe. A change inthe voltage signal may correspond to a location along the hybridizedbiopolymer containing the probe. The voltage signal may be processedusing a computer algorithm to reconstruct the sequence of thebiopolymer. The biopolymer may include or consist essentially of adouble-stranded biopolymer target molecule. Preparing the analyte mayinclude contacting the target molecule with a first probe having a firstprobe specificity for recognition sites of the target molecule to form afirst plurality of local ternary complexes, the first probe having afirst predicted recognition site sequence. The voltage signal may beused to determine positional information of the first plurality of localternary complexes. The positional information may include a parameter toa spatial distance between two local ternary complexes.

Preparing the analyte may further include contacting the target moleculewith a second probe having a second probe specificity for recognitionsites of the target molecule to form a second plurality of local ternarycomplexes, the second probe having a second predicted recognition sitesequence.

The voltage signal may be used to determine positional information ofthe second plurality of local ternary complexes. Positional informationof at least the first and second plurality of local ternary complexesmay be aligned to determine a DNA sequence of the target.

The biopolymer may include or consist essentially of a double-strandednucleic acid target molecule having a plurality of binding sitesdisposed along the sequence thereof. Preparing the analyte may includeadding a plurality of probe molecules having a first sequencespecificity to the double stranded nucleic acid target molecule.

The probe molecules having the first sequence specificity and the targetmolecule may be incubated so as to effectuate preferential binding ofthe first probe molecules to both a first binding site and a secondbinding site of the target molecule. The voltage signal may be used tomeasure a parameter related to a distance between the first binding siteand the second binding site.

Preparing the analyte may include contacting the biopolymer with a firstprobe to create at least one probe-target complex at a recognition siteof the biopolymer for which the first probe has a known specificity,while leaving uncomplexed, regions of the biopolymer for which the firstprobe is not specific. Preparing the analyte may include contacting thebiopolymer with a second probe to create at least one probe-targetcomplex at a recognition site of the biopolymer for which the secondprobe has a known specificity, while leaving uncomplexed, regions of thetarget for which the second probe is not specific.

The voltage signal may be used to detect and record complexed anduncomplexed regions of the biopolymer to create a first probe map of thefirst probe and a second probe map of the second probe, the first probemap and the second probe map incorporating information on the relativeposition of the hybridization of the probes. A candidate sequence may bedetermined by aligning at least two probe sequences using positionalinformation or a combination of overlapping sequences of the probemolecules and positional information. The first and second probe mapsmay incorporate information on an error of the positional informationfor each probe. A candidate sequence may be determined by ordering atleast two probe sequences using positional information and parametersrelating to the error in positional information or a combination ofoverlapping sequences of the probe molecules and positional informationand error in positional information.

The description of elements of the embodiments of other aspects of theinvention may be applied to this aspect of the invention as well.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic diagram illustrating a longitudinally displacedtransverse electrode device configuration in accordance with anembodiment of the invention;

FIG. 2 is a schematic diagram illustrating a longitudinally displacedtransverse electrode device configuration in accordance with anotherembodiment of the invention;

FIG. 3 is a schematic diagram illustrating a longitudinally displacedcontinuous transverse nanoscale electrode device configuration inaccordance with another embodiment of the invention;

FIG. 4 is a schematic diagram illustrating a longitudinally displacednano scale electrode device configuration with electrodes disposed onthe same side of a nanochannel, in accordance with an embodiment of theinvention;

FIG. 5 is a schematic depiction of a DNA molecule;

FIG. 6 is a schematic depiction of an RNA molecule;

FIG. 7 is a schematic depiction of a hybridizing oligonucleotide (orprobe);

FIG. 8 is a schematic depiction of a single strand DNA moleculehybridized with a probe; and

FIG. 9 is a graph of an exemplary voltage signal determined inaccordance with an embodiment of the invention.

DETAILED DESCRIPTION Fabrication of Fluidic Channel and SensingElectrodes

Embodiments of the invention include devices and methods for performingsequence analysis by hybridization (“SBH”). Referring to FIG. 1, in anembodiment a device 100 includes a fluidic channel 105, e.g., a micro-or nanochannel. A pair of electromotive electrodes 110, 110′ aredisposed at a first and a second end of the fluidic channel 105 forapplying a potential along the fluidic channel. A pair of sensingelectrodes 115A, 115B are disposed at two discrete locations along alength of the fluidic channel. The sensing electrodes may be inelectrical communication with a measurement tool 120 that measures avoltage sensed by the sensing electrodes. The fluidic channel 105 may bedefined in a substrate comprising either silicon, silicon dioxide, fusedsilica, or gallium arsenide, and may contain an electrolytic solution.The electromotive electrode pair 110, 110′ may include at least oneanode 110′ and cathode 110′ in contact with the electrolytic solution toprovide a constant or changing current to drive the analyte 125 throughthe fluidic channel 105. In an alternate embodiment, a pressuredifferential, such as a positive pressure, may be used to drive theanalyte 125 through the fluidic channel 105. Pressure may be suppliedwith a fluid pump or with a pressurized gas line. Other methods ofapplying pressure may be envisioned by one of skill in the art. In someembodiments, a chemical potential gradient may be used to move moleculesthrough the fluidic channel 105. The analyte may also be translocated byusing a chemical potential gradient. Chemical potential gradients may becreated with concentration gradients. For instance, a fluidic channelmay have one end immersed in a fluid that has a higher saltconcentration than the fluid at the other end of the channel. Thedifferential in salt concentration at the ends of the fluidic channelcauses an osmotic pressure that can drive analytes through the channel.

As the analyte 125, which may be any biopolymer including, but notlimited to, polypeptides, DNA or RNA, passes through the fluidic channel105, it will pass between the pair of sensing electrodes 115A, 115B(each individually referred to herein as “A” and “B”). The sensingelectrodes 115A, 115B contacting the fluidic channel 105 may be used tomeasure the changes in conductance of the electrolytic volume betweenthem. The changes in conductance between the sensing electrodes 115A,115B may be measured using a measurement tool 120, e.g., a voltmeter.

By making the longitudinal distance, e.g., a length along the fluidicchannel 105, between sensing electrodes 115A, 115B small, the device 100retains high sensitivity for an analyte 125 passing therethrough. Eachsensing electrode 115A, 115B in the pair may be disposed on oppositesides of the fluidic channel 105 as in FIG. 1, where tips of the sensingelectrode 115A, 115B are in contact with the electrolytic solution areentirely or partially across from one another, or as in FIG. 2, in whichthe tips of the sensing electrodes 115A, 115B are not across from eachother, but are rather longitudinally displaced with respect to oneanother by a selected distance. Alternatively, each sensing electrode115A, 115B in a pair may cross the fluidic channel 105, as shown in FIG.3. Referring to FIG. 4, in a third arrangement, two sensing electrodes115A, 115B in a pair may be on the same side of the fluidic channel 105.

The devices 100 described herein may be formed by the fabrication of atrench to define a fluidic channel 105 having nanoscale dimensions, andthe fabrication of nanoscale electrodes. A typical device 100 may alsohave a microscale fluidic structure for introduction of buffers andsamples. Thus, the techniques described herein employing nanochannelsare also applicable to devices including microchannels. Some or all ofthe structures may also be sealed with a cap in order to provide closedchannels.

Fluidic channels may be formed in the substrate by, e.g., lithographicand etch steps. The substrate may be, e.g., a silicon-on-insulatorwafer, with, for example, an (100) Si surface, a Si wafer, or a fusedsilica substrate. Lithography in the sub-100 nanometer (nm) regime maybe performed by various techniques, including the following: electronbeam lithography (EBL), nanoimprint lithography (NIL) or deepultraviolet optical lithography (DUV OL). See Liang, X.; Morton, K. J.;Austin, R. H.; Chou, S. Y., Single sub-20 nm wide, centimeter-longnanofluidic channel fabricated by novel nanoimprint mold fabrication anddirect imprinting, Nano Lett. 2007, 7, 3774-3780; Austin, M. D.; Ge, H.;Wu, W.; Li, M.; Yu, Z.; Wasserman, D.; Lyon, S. A.; Chou, S. Y.,Fabrication of 5 nm line width and 14 nm pitch features by nanoimprintlithography, App. Phys. Lett. 2004, 84, 5299-5301; and Guo, J., Recentprogress in nanoimprint technology and its applications, J. Phys. D:Appl. Phys. 2004, 37, R123-R141. Each of these references isincorporated herein by reference in its entirety. The current industrystandard in micro and nanofabrication is optical lithography due to itslow cost and high throughput. At present, optical lithography has beensuccessfully used in the mass production of devices with criticaldimensions as small as 32 nm. EBL and NIL are presently used extensivelyin academic research environments due to their versatility andcapability of producing sub-10 nm features reproducibly. Any of thesemethods may be used to pattern the fluidic trenches described herein.

The removal of material for the formation of the fluidic trenches may beperformed by, e.g., etching. Wet etching includes the immersion of thematerial in a solution capable of selective removal. Dry etching, i.e.,reactive ion etching (RIE), involves the exposure of the sample to acharged plasma. For the resolution and control required of nanoscalefabrication, RIE is preferable due to its consistency, controllability,and efficiency. Microfluidic channels or reservoirs leading to thenanoscale channels may be etched using either wet or dry methods.

The resulting channels have preferred dimensions of width and depthranging from 1 nm to 5 μm, more preferably 1 nm to 1 μm, and morepreferably 10 nm to 100 nm. The channels may have a length selected froma range of, e.g., 1 micrometer (μm) to 10 centimeters (cm).

The size of the channel may be chosen with regard to the persistencelength of the analyte. For example, a randomly coiled polymer (e.g.,DNA) may be elongated when introduced into a confined space, such thatwhen the confinement space becomes smaller the extent of elongationbecomes greater. In some embodiments, it may be preferable to elongatethe analyte to measure length or distance between probes. Depending onthe cross-sectional size and the persistence length it may be useful tohave the geometric mean of the width and depth of the channel be between5% and 500% of the persistence length of the analyte. For example, fordouble-stranded DNA, under conditions where the persistence length is 50nm, it may be preferable to have, e.g., a fluidic channel with a widthand depth between 2.5 nm and 250 nm. In other embodiments, for morerigid polymers such as RecA coated DNA, under conditions where thepersistence length is 950 nm, it may be preferable to have, e.g., afluidic channel with a width and depth between 45 nm to 4.75 μm.

After the channels are formed, sensing electrodes are fabricated.Similar to etching and lithography, numerous metal deposition techniquessuitable for fabrication of sensing electrodes exist in conventionalmicrofabrication process flows. Each technique has positive and negativeattributes and a list of the materials that may be deposited using thattechnique. The three primary techniques are: electron beam evaporation,thermal evaporation, and sputtering. The sensing electrodes havethicknesses ranging from 5 nm to 100 nm at the point where the sensingelectrodes intersect the fluidic channels. The sensing electrodes may bewider and/or thicker in regions distal to the fluidic channels andapproaching contact pads disposed at the perimeter of the device.

To complete the device, a cap layer may be introduced to preventevaporation of liquid from the fluidic channel. The cap may be formedover just the nanoscale fluidic paths or over all of the fluidicchannels. In the latter case, the cap structure preferably has holes orports to allow for the introduction of fluid and samples into thefluidic paths. In another embodiment, the entire substrate, i.e., wafer,may be capped. The cap may be made of a glass plate such as borosilicateglass, phosphosilicate glass, quartz, fused silica, fused quartz, asilicon wafer or other suitable substrates. Various techniques aresuitable for accomplishing this step including anodic bonding. In anodicbonding, an underlying silicon wafer and a glass substrate are pressedtogether and heated while a large electric field is applied across thejoint. Anodic bonding has been demonstrated to form a strong bondbetween a silicon wafer and the capping substrate. Direct siliconbonding has been used to join two silicon wafers. The latter methodinvolves pressing the two wafers together under water. Other methods usean adhesive layer, such as a photoresist, to bond the cap to thesubstrate.

An exemplary fabrication process for defining the proposed sensingelement is as follows. A suitable substrate, such as a conventional(100) p-type silicon wafer, is thermally oxidized in a hydratedatmosphere to grow a thick (e.g., >1 μm) silicon-dioxide (SiO₂) layer.This SiO₂ layer may serve as insulation between subsequently formedadjacent metal sensing electrodes, and may also reduce overall devicecapacitance.

Using conventional high resolution optical lithography, the pattern ofthe fluidic channel may be transferred to a first photoresist maskinglayer. RIE with an anisotropic etch species, such as Cl₂, may be used totransfer the pattern into the SiO₂ layer. The preferred width and depthof the channel may be determined by the requirements for the devicesensitivity. The smaller the volume of the channel between two sensingelectrodes, the more sensitive the device is. Channel size, width, anddepth, may also be determined by the size or behavior of the analyte. Inone embodiment, the device described herein is used to detect strands ofDNA. It may be desirable to fabricate the channel with dimensions thatextend the DNA strand within the channel. For instance fordouble-stranded DNA, it has been found that the use of channels withdimensions of 100 nm or less are able to extend the biopolymer. SeeTegenfeldt, J. O et al. The dynamics of genomic-length DNA molecules in100-nm channels. Proc. Nat. Acad. Sci. USA, 2004, 101, 10979-10983,which is incorporated herein by reference in its entirety. Uponcompletion of the dry etch procedure, residual resist is removed and thesubstrate vigorously cleaned.

Following the etching of the fluidic channel, embedded metal sensingelectrodes are fabricated. Conventional high resolution opticallithography may be used to transfer the metal electrode pattern to asecond photoresist masking layer. RIE with an anisotropic etch species,such as Cl₂, will be used to transfer the pattern into the SiO₂ layer.Preferably the depth of these trenches exceeds or equals the depth ofthe fluidic channel. Upon completion of pattern transfer to the SiO₂layer, a thin metal adhesion promotion layer may be deposited. Asuitable layer is tantalum with a thickness of 30-50 Å, deposited viaelectron beam evaporation. Next, the sensing electrode material isdeposited without exposing the substrate to atmosphere. A preferredmetal for the bulk of the sensing electrodes is platinum, also depositedvia electron beam evaporation. Other examples of suitable metals includegold, chrome, titanium, silver chloride, silver, and graphene. Thethickness of the metal is dictated by the depth of the etched trenches,such that the resultant metal trace is approximately planar with a topsurface of the SiO₂ layer. Upon completion of the metal deposition, thesubstrate is immersed in a photoresist solvent that will lift-off excessmetal from the surface and the substrate is vigorously cleaned.Chemical-mechanical polishing (CMP) may be performed to remove excessmetal extending over the SiO₂ top surface, thereby planarizing a topsurface of the metal to be level with the SiO₂ top surface.

To complete the fabrication of the sensor, a cap layer is preferablyadhered to the sensor surface to provide a leak-free seal, enablingfluidic conduction. Preferred cap materials include borosilicate glass,fused silica, fused quartz, quartz, or phosphosilicate glass. Holes maybe created in the cap layer to provide access to fluidic inlet, fluidicoutlet and metal sensing electrodes. A typical method for making holesin glass wafers is ultrasonic etching, which allows for highlycontrollable pattern transfer to glass substrates. Anodic bonding maythen be used to bond the glass cap layer to the underlying substrate,e.g., silicon wafer. The anodic bonding of two layers provides a strongand leak-free seal.

An exemplary device with a pair of such nanoscale sensing electrodes115A, 115B is illustrated in FIG. 1, i.e., electrodes A and B. Electriccurrent is transferred in the form of ionic flow in an electrolytesolution confined in the fluidic channel 105, e.g., a nanochannel. Therole of the electrolyte is to maintain a uniformly distributed electricfield in the fluidic channel. Typical electrolyte solutions have beendescribed in applications of electrophoresis to separations of DNAmolecules. The most common electrolytes for electrophoretic separationof DNA are Tris boric acid EDTA (TBE) and tris acetate EDTA (TAE). See,e.g., Sambrook, J.; Russell, D. W. Molecular Cloning: A LaboratoryManual 3^(rd) ed. Cold Spring Harbor Press, 2001. However, anyconductive medium may be used.

Operation of Device

During operation, a constant current is supplied by applying a potentialto a pair of macroscopic electrodes, e.g., electromotive electrodes 110,110′ disposed at opposing ends of the fluidic channel 105 and in contactwith the electrolytic solution. The electromotive electrodes arepreferably in electrical communication with the wires leading to theends of the fluidic channel 105 illustrated in FIGS. 1-4. A potentialmay be applied along the fluidic channel 105 to generate anelectrophoretic force therein, such that the analyte 125 is translocatedfrom a first end of the fluidic channel 105 to a second end of thefluidic channel. The electromotive electrodes may generate a constant oroscillating electrophoretic force in the fluidic channel 105 fortranslocation of an analyte 125 disposed therein. The voltage betweenthe electromotive electrodes may be constant or it may be changed overthe course of a measurement. For instance, it may be desirable to reducethe voltage once a DNA molecule has entered the fluidic channel 105 andbefore the DNA molecule has entered the volume between the sensingelectrodes, in order to slow the passage of the DNA molecule through thevolume between the sensing electrodes. Controlling the rate of passageof the DNA molecule through the volume between sensing electrodes 115A,115B allows for more versatile detection of the DNA.

As an example of the placement of sensing electrodes, a width of 20 nmmay be assumed for each of sensing electrodes A and B in FIG. 1.Electrode A may be shifted along the fluidic channel 105 relative toelectrode B, by, e.g., 10 nm or 30 nm. Distances between sensingelectrodes from 30 nm to 100 nm or from 30 nm to 500 nm, or from 30 nmto 5 μm can be incorporated into a single device. For analytes ofsufficient length, distances up to e.g., 500 μm may be used, e.g., up to300 μm, 200 μm, or 100 μm may be used. Although electrodes with anydistance therebetween may be fabricated, since DNA is difficult toobtain at a length greater than 500 μm, any electrode distance that isgreater than 500 μm would be superfluous, as long as the length of theDNA does not exceed 500 μm. The smaller displacement between electrodesA and B is an example of an embodiment in which there is overlap of theelectrodes, even though they are displaced with respect to one another.In some embodiments, as shown in FIG. 2, there may be no overlap betweensensing electrodes 115A, 115B.

The voltage across sensing electrodes 115A, 115B is proportional to thelocal impedance in the fluidic channel 105 between sensing electrodes115A, 115B. The spacing of the electrodes is determined by severalfactors. The smaller the distance between electrodes in a sensing pair,all other factors being constant, the smaller the particle that can bedetected by the sensing pair. However, fabrication limits may make itdifficult to consistently place the electrodes in a pair at smalldistances. Thus, the selected distance is a trade-off betweenfabrication reproducibility and sensitivity of the device 100. Thechoice of separation distance and thus whether the electrodes areoverlapping or non-overlapping depends on these constraints.

The resulting sensing electrode 115A, 115B arrangement provides a meansto separate the current and voltage probes and can be used to employ4-point sensing in a fluidic channel. In an embodiment, the macroscopic,electromotive electrodes 110, 110′ at the ends of the fluidic channel105 provide a current while the nanoscale sensing electrodes 115A, 115Bdisposed across the fluidic channel 105 are used to measure voltage. Thevoltage electrodes preferably have an output impedance higher than theimpedance of the volume being measured.

The following calculations demonstrate the feasibility of this deviceconcept. The fluidic channel may be subject to a constant electric fieldequal to the potential difference along the length of the channeldivided by the length of the channel, i.e., 100 mV appliedlongitudinally to a 10 μm long fluidic channel results in a field of 100mV/10 μm=10 mV/μm or 0.01 mV/nm. The potential difference betweenelectrodes A and B separated by 10 nm is then the product of thedistance between electrodes and the electric field or:

10 nm×0.01 mV/nm=0.1 mV.

Similarly, a potential difference of 0.3 mV exists between electrodes Aand B when the spacing is 30 nm. Each of these potentials is readilydetectable with conventional electronic measurement tools. When a DNAmolecule or any other analyte passes between a pair of sensingelectrodes, the impedance between the sensing electrodes changes due toa resistivity difference between the electrolyte and the molecule. Theresulting transient change in the potential is measured, whilemaintaining a constant current.

For the example shown in FIG. 1, assuming a substantially constantvelocity, the duration of each voltage pulse detected by the sensingelectrodes 115A, 115B is proportional to the length of the DNA or otheranalyte 125 that passes between the two sensing electrodes. Afterdetermining the speed of the analyte 125 in the fluidic channel, themeasured duration of the pulse may be used to calculate the distance theanalyte 125 moved (velocity×time=distance) while passing through thevolume between sensing electrodes, which would be equal to the analyte's125 length.

It is important to note that by shifting one of the transverseelectrodes along the fluidic channel 105 by a distance of 10-50 nm, andusing a fluidic channel 105 with a diameter of about 10 nm, the volumeseparating the two sensing electrodes 115A, 115B may be viewed as havinga sensitivity equivalent to that of a conventional solid-state nanopore.

In use, the voltage between a pair of sensing electrodes 115A, 115B,e.g., V_(AB), may be sensed by a measurement tool 120, e.g., avoltmeter, configured to measure the potential difference between thesensing electrode 115A, 115B pair. In a preferred embodiment, thevoltmeter 120 may be in electrical communication with each of thesensing electrodes 115A, 115B in the pair via metal contact padsconnected to nanowires leading to the sensing electrodes.

Generally, an analyte 125 may be detected in the fluidic channel 105 asfollows. The analyte, e.g., the biopolymer strand and probes, istransferred from a chamber into the fluidic channel in an electrolyticsolution. Typically, an electrolyte may be added to the fluidic channelby a pipette, a syringe, or a pump. An analyte sample size may be assmall as practically possible, as the device allows the detection ofsingle molecules. The fluid may wet the fluidic channels by capillaryaction. Analyte may be introduced into the microscale areas either withthe original electrolyte or after by pumping in a new solution. Ananalyte, such as DNA, which may be hybridized to one or more probes, maybe drawn into the fluidics channel by the potential. For small analytes,one could use diffusion, fluid flow, or a potential.

The fluidic channel may have a width that is no smaller thanapproximately the same width as the analyte, and may be sufficientlylarge such that large molecules bound to the analyte may pass throughthe fluidic channel. For example, the width of the fluidic channel maybe selected from a range of 1 nm to 200 nm. The fluidic channel may besufficiently deep to allow large molecules bound to the analyte to passthrough and yet shallow enough to be approximately the same size as theanalyte. The fluidic channel depth may be, e.g., selected from a rangeof 1 nm to 200 nm. The length of the fluidic channel may be selectedsuch that the entire analyte is contained in the fluidic channel.

In an embodiment, the sensing electrodes and fluidic channel may bepreferably arranged such that the entire analyte enters the fluidicchannel before it enters the volume between sensing electrodes. Thisconfiguration provides an advantage of reducing the effect of theanalyte on the conductance of the fluidic channel. For instance, if oneis beginning to measure the change in potential of a volume betweensensing electrodes while the conductance of the whole fluidic channel ischanging due to more analyte entering the fluidic channel, the analysisbecomes more complex In a preferred embodiment, the analyte may becontained completely in the channel when it exits the volume betweensensing electrodes. Thus, the length of the fluidic channel preferablyhas a minimum length that is approximately three times the length of theanalyte (assuming that the volume between sensing electrodes is only aslong as the analyte, which is a minimal requirement but not optimal).The length of a 1 kb piece of DNA is about 330 nm, so a length of thefluidic channel is preferably at least 1 μm in length. The longest pieceof DNA suitable for analysis with the described methods may be 10megabases (Mb), which corresponds to a preferred fluidic channel of atleast 10 mm. More preferably, the length of a fluidic channel is tentimes the length of the analyte, and thus a more preferred upper limitfor a channel length is 100 mm (10 cm) Thus, the fluidic channel lengthis preferably selected from a range from 1 μm to 10 cm. Longer andshorter fluidic channel lengths are also possible.

In some embodiments, the structure of the fluidic channel may facilitateentry of the analyte into the channel, e.g., the fluidic channel maycomprise a series of posts (e.g., U.S. Pat. No. 7,217,562, which isincorporated by reference in its entirety) and/or a funnel shape.

The analyte is translocated through the fluidic channel by a currentthat is supplied by applying a potential to the two electromotiveelectrodes disposed at opposing ends of the fluidic channel and incontact with the electrolytic solution. The electromotive electrodes maygenerate a constant or oscillating electrophoretic force in the fluidicchannel for translocation of an analyte disposed therein. The voltagebetween the macroscopic electrodes may be constant or it may be changedover the course of a measurement. For example, the voltage may bereduced once a DNA molecule has entered the fluidic channel and beforethe DNA molecule has entered the volume between the sensing electrodes,to slow the passage of the DNA molecule through the volume betweensensing electrodes.

A voltage signal reflecting a change in potential between the pair ofsensing electrodes may be monitored. As the analyte moves through avolume between the sensing electrodes, the voltage signal changes. Thesignal may be elevated or depressed for a period of time that reflectsthe length of the analyte, e.g., a probe-target complex, or the lengthof the intervening regions without probes. A typical analyte isnon-conductive and will impede the flow of ions in the electrolyte.Therefore, the potential—and voltage signal—typically increase as theanalyte flows through the volume between sensing electrodes. In someembodiments, e.g., a low salt electrolyte and a charge-carrying analyte,the potential and voltage signal may decrease as the analyte flowsthrough the volume between sensing electrodes. The voltage signalfurther changes when the portion of the analyte containing thehybridized probe moves through the volume between the sensingelectrodes.

Determination of Analyte Length and Probe Location

In an embodiment, a method for detecting the relative position of probeshybridized to a biopolymer and/or the length of the biopolymer.Nanopores may be used as detectors to determine the distance betweenhybridization sites as described in U.S. Patent Publication No.2007/0190542 A1, which is incorporated herein by reference in itsentirety. The construction of a nanochannel device incorporating voltagedetectors is described herein. In both the nanopore and the fluidicchannel (e.g., a nanochannel), the distance between hybridization siteson the target biopolymer may be inferred from the time between thedetection of a first hybridization position and a subsequenthybridization position as the biopolymer moves through the nanopore orfluidic channel. The technology disclosed herein allows thedetermination of biopolymer length and distances between hybridizationpositions.

In particular, as used herein, a “probe” means any molecule or assemblyof molecules capable of sequence-specific covalent or non-covalentbinding to a target molecule. A probe may be, but is not limited to, aDNA sequence, an RNA sequence, antibodies or antibody fragments. Theterms “nucleotide” and “base” are used interchangeably and mean amolecule consisting of a phosphate group, a sugar and one of fivenitrogen-containing bases that can make up DNA or RNA polynucleotidechains or strands. For DNA, the nitrogen-containing bases includecytosine (C), adenine (A), guanine (G) and thymine (T) and the sugar isa 2-deoxyribose. For RNA, a the deoxyribose sugar is replaced by aribose sugar instead of deoxyribose and uracil bases (U) instead ofthymine bases (T).

A DNA probe “library” is a collection of DNA probes of a fixed lengthwhich includes a large number of, or possibly all, possible sequencepermutations. A plurality of probes may be made up of multiple copies ofthe same probe with the same sequence selectivity or be made up of twoor more probes with different sequence selectivity. A “probe map” meansa data set containing information related to the sites along a targetsequence at which a probe preferentially binds. A partially hybridizedbiomolecule is created when the entire length of a sequence selectiveprobe binds to a portion of the length of the target biomolecule. Thedata set may include absolute positional information referenced to aknown sequence, relative information related to distances betweenbinding sites, or both. The data set may be stored in computer media.Further details of the characteristics of probe and spectrum maps may befound in U.S. Patent Publication No. 2009-0099786 A1, which isincorporated herein by reference in its entirety.

A “target,” i.e., the analyte, is a biopolymer, of which length,identity or sequence information is to be determined using embodimentsof the present invention. The analyte may be a biopolymer, such as adeoxyribonucleic acid, a ribonucleic acid, proteins, or a polypeptide.The target DNA may be single- or double-stranded. In some embodiments,the analyte is a biopolymer to which probes have been hybridized.

DNA is the fundamental molecule containing all of the genomicinformation required in living processes. RNA molecules are formed ascomplementary copies of DNA strands in a process called transcription.Proteins are then formed from amino acids based on the RNA patterns in aprocess called translation. The common relation that can be found ineach of these molecules is that they are all constructed using a smallgroup of building blocks, such as bases or amino acids, that are strungtogether in various sequences based on the end purpose that theresulting biopolymer will ultimately serve.

Analytes may be prepared for analysis, e.g., as disclosed in U.S. PatentPublication No. 2007/0190542, which is incorporated herein by referencein its entirety. Referring to FIG. 5, a DNA molecule 500 isschematically depicted and can be seen to be structured in two strands505, 510 positioned in anti-parallel relation to one another. Each ofthe two opposing strands 505, 510 is sequentially formed from repeatinggroups of nucleotides 515 where each nucleotide 515 consists of aphosphate group, 2-deoxyribose sugar and one of four nitrogen-containingbases. The nitrogen-containing bases include cytosine (C), adenine (A),guanine (G) and thymine (T). DNA strands 505 are read in a particulardirection, from the so called the 5′ or “five prime” end to the socalled the 3′ or “three prime” end. Similarly, RNA molecules 600, asschematically depicted in FIG. 6 are polynucleotide chains, which differfrom those of DNA 500 by having ribose sugar instead of deoxyribose anduracil bases (U) instead of thymine bases (T).

Traditionally, in determining the particular arrangement of the bases515 in these organic molecules and thereby the sequence of the molecule,a process called hybridization is utilized. The hybridization process isthe coming together, or binding, of two genetic sequences with oneanother. This process is a predictable process because the bases 515 inthe molecules do not share an equal affinity for one another. T (or U)bases favor binding with A bases while C bases favor binding with Gbases. This binding is mediated by the hydrogen bonds that exist betweenthe opposing base pairs. For example, between an A base and a T (or U)base, there are two hydrogen bonds, while between a C base and a G base,there are three hydrogen bonds.

The principal tool that is used then to determine and identify thesequence of these bases 515 in the molecule of interest is a hybridizingoligonucleotide commonly called a probe 700. As FIG. 7 illustrates, aDNA probe 700 is a DNA sequence having a known length and sequencecomposition. Probes 700 may be of any length dependent on the number ofbases 515 that they include. For example a probe 700 that includes sixbases 515 is referred to as a six-mer wherein each of the six bases 515in the probe 700 may be any one of the known four natural base types A,T(U), C or G and alternately may include non-natural bases. In thisregard the total number of probes 700 in a library is dependent on thenumber of bases 515 contained within each probe 700 and is determined bythe formula 4^(n) (four raised to the n power) where n is equal to thetotal number of bases 515 in each probe 700. Accordingly, the generalexpression for the size of the probe library is expressed as 4^(n) n-merprobes 700. For the purpose of illustration, in the context of a six-merprobe the total number of possible unique, identifiable probecombinations includes 4⁶ (four raised to the sixth power) or 4096 uniquesix-mer probes 700. It should be further noted that the inclusion ofnon-natural bases allows the creation of probes that have spaces orwildcards therein in a manner that expands the versatility of thelibrary's range of probe recognition. Probes that include universalbases organized into patterns with natural bases may also be used, forexample those described in U.S. Pat. Nos. 7,071,324, 7,034,143, and6,689,563, each of which are incorporated herein by reference in theirentireties.

When a target biomolecule, such as single-stranded DNA, is incubatedwith a sequence selective probe under appropriate conditions, the probehybridizes or binds to the biomolecule at specific sites. Thedetermination of the relative location of the hybridization sites isuseful for constructing maps of the target biomolecule, and foridentifying the target molecule.

When the biopolymer to be analyzed is a double-stranded DNA 500, theprocess of hybridization using probes 700 as depicted in FIG. 8 firstrequires that the biopolymer strand be prepared in a process referred toas denaturing. Denaturing is a process that is usually accomplishedthrough the application of heat or chemicals, wherein the hydrogen bondsbetween the two strands of the original double-stranded DNA are broken,leaving a single strand of DNA whose bases are available for hydrogenbonding. After the biopolymer 800 has been denatured, a single-strandedprobe 700 is introduced to the biopolymer 800 to locate portions of thebiopolymer 800 that have a base sequence that is complimentary to thesequence that is found in the probe 700. In order to hybridize thebiopolymer 800 with the probe 700, the denatured biopolymer 800 and aplurality of the probes 700 having a known sequence are both introducedto a solution. The solution is preferably an ionic solution and morepreferably is a salt containing solution. The mixture is agitated toencourage the probes 700 to bind to the biopolymer 800 strand alongportions thereof that have a matched complementary sequence. Once thebiopolymer strand 800 and probes 700 have been hybridized, the strand800 is introduced to one of the chambers of a sequencing arrangement. Itshould also be appreciated to one skilled in the art that while thehybridization may be accomplished before placing the biopolymer strand800 into the chamber, it is also possible that the hybridization may becarried out in one of these chambers as well. In this case, after thedenatured biopolymer has been added to the cis chamber, a drop of buffersolution containing probes 700 with a known sequence are also added tothe cis chamber and allowed to hybridize with the biopolymer 800 beforethe hybridized biopolymer is translocated.

As a specific example of analyte preparation, a nucleotide sample, suchas DNA or RNA, may be heated to a denaturing temperature, typicallygreater than 90° C. for DNA and typically between 60-70° C. for RNA, inthe presence of a selection of probes and in a buffer of 50 mM potassiumchloride and 10 mM Tris-HCl (pH 8.3). The hybridization may beaccomplished in the cis chamber or before placing the analyte in thechamber. The mixture of nucleotide strand and probes are then cooled toallow for primer binding, with the temperature dependent upon the lengthand composition of the probe. For example, a 6-nucleotide probe would becooled to room temperature or lower. The nucleotide strand and probe maybe allowed to anneal at the low temperature for up to 5 minutes beforebeing passed through a fluidic channel for analysis. The exacttemperatures may be easily determined by one of skill in the art withoutundue experimentation.

Referring to FIG. 1, in some embodiments, electrical signalscorresponding to the volume between sensing electrodes 115A, 115B aredetected by the sensing electrodes 115A, 115B as an analyte 125, e.g., abiopolymer, is disposed in the fluidic channel. As the biopolymer entersthe volume between sensing electrodes, a change in the electrical signalis recorded. Referring to FIG. 10, the electrical signal increaseslinearly at time point T₁ as the analyte enters the volume betweensensing electrodes. When the volume between sensing electrodes iscompletely filled, the signal stays essentially constant. When theanalyte leaves the volume between sensing electrodes, the signal returnsto baseline at time point T₄. The sensing electrodes are configured forconnection to a measurement tool for capturing the electrical signalscorresponding to the volume between sensing electrodes, e.g., avoltmeter. Electrical signals captured by the measurement tool may berecorded as a function of time by a data collection device, e.g., aStanford Research Instruments SIM970 voltmeter. A time interval betweenvoltage signal changes may be recorded. The duration of the change inthe voltage signal over baseline may indicate a presence of an analyte.Another increase over that, as shown in FIG. 9 between time points T₂and T₃, may indicate the presence of a probe hybridized with theanalyte. The electrical signal may have fluctuations from noise and fromthe fact that a long analyte, such as DNA, typically has small bends init. When a section having a bend enters the volume between sensingelectrodes, the electrical signal may increase slightly, and then maydecrease slightly when the bend exits the volume between sensingelectrodes. Calibration of the electrical signals, therefore, may beneeded to determine length. To determine the length of the analyte, onemay calibrate the system with one or more standards, e.g., biopolymersof known, varying lengths, to determine the pulse duration and speed ofeach biopolymer length. Under consistent voltage and electrolyteconditions, this data may be used to create a standard curve forcorrelating the pulse duration of the analyte with its length.

Similarly, the duration of a change in the voltage signal may be used todetermine the location of hybridization of a first plurality of probesand a distance between two probes on the biopolymer. The detectedelectrical signal corresponding to volume between sensing electrodes ofthe fluidic channel may be detected by using the sensing electrodes. Asshown in FIG. 9 at time point T₁, the electrical signal may initiallychange when the biopolymer moves through a volume between sensingelectrodes and further change, at T₂, when a portion of the biopolymerincluding a hybridized probe moves through the volume between sensingelectrodes. The detected electrical signals may indicate the locationsof the hybridized probes along the biopolymer. Changes and duration ofchanges in electrical signals may be analyzed to determine the locationof probes with known sequence specificity along the length of thebiopolymer. Distances between probes may be based upon the durationbetween voltage spikes representing hybridized probes just as theduration of spike is used to determine biopolymer length. For example,one may consider the average of the voltage signals when determining thedistance between hybridized probes. Outliers may be excluded if theydeviate greatly from other measurements taken of the same molecule. Thisanalysis may be done either visually or with the assistance of acomputer program that executes the analysis described herein.

The calculation of distances between probes may be used to determine thesequence of a biopolymer as follows. An analyte may be prepared byhybridizing a first plurality of probes with a known sequence with thebiopolymer such that the first plurality of probes attaches to portionsof the biomolecule to produce a partially hybridized biomolecule. Theanalyte may be disposed in a fluidic channel. A potential may be appliedalong the fluidic channel to generate an electrophoretic force thereinsuch that the analyte is translocated from a one end of the fluidicchannel to another end of the fluidic channel. Changes in the voltageare used to detect the hybridized probe as described above.

At least a portion of the sequence of the biopolymer may be determinedby detecting the hybridization of the first plurality of probes. Itslocation on the biopolymer may be determined by using a distance fromthe end of the biopolymer to a probe's site of hybridization or thedistance from a probe site of hybridization to another probe site ofhybridization. A computer algorithm may be used to process theelectrical signals to help determine the sequence of the biopolymer.

In some embodiments, a second plurality of probes having specificity forrecognition sites on the target molecule may be hybridized with thebiopolymer, either subsequently or in parallel to the first probe, toform individual pluralities of hybridization and the detecting,analyzing, and determining steps may be repeated with the subsequentplurality of probes.

The biopolymer may include a double-stranded biopolymer target molecule.The analyte may be prepared by contacting the biopolymer, i.e., thetarget molecule, with a first probe having a first probe specificity forrecognition sites of the target molecule to form a first plurality oflocal ternary complexes.

The electrical signals may be used to detect and record complexed anduncomplexed regions of the biopolymer to create a first probe map of thefirst plurality of probes and subsequent probe maps for each subsequentplurality of probes, the first probe map and subsequent probe maps eachincluding information about the relative positions of the hybridizedfirst and each of the subsequent plurality of probes. Each probe map mayinclude a series of numbers that indicate the distances between probes.The numbers may indicate distance in terms of base pairs or distance interms of nanometers. A candidate sequence for at least a portion of thebiopolymer may be determined by ordering at least two probe sequencesusing positional information and/or a combination of overlapping probebinding sequences and positional information.

The first and second probe maps may include information about an errorof the positional information for each probe. For example, eachindicated distance may have an associated standard deviation, e.g., 100nm±10 nm. Further, a candidate sequence may be determined by ordering atleast two probe sequences using at least one of (i) positionalinformation and parameters relating to the error in positionalinformation or (ii) a combination of overlapping sequences of the probemolecules and positional information and error in positionalinformation.

The sequencing of biopolymers by hybridization of probes to form ternarycomplexes is further discussed in patent application U.S. Ser. No.12/243,451 which is incorporated herein by reference in its entirety.Additional background information about detection/sequencing may befound in Gracheva, M. E.; Xiong, A.; Aksimentiev, A.; Schulten, K.;Timp, G, Leburton, J.-P. Simulation of the electric response of DNAtranslocation through a semiconductor nanopore-capacitor, Nanotechnology2006, 17, 622-633; and Zwolak, M.; Di Ventra, M. Physical approaches toDNA sequencing and detection, Rev. Mod. Phy. 2008, 80, 141-165, each ofwhich is incorporated herein by reference in its entirety.

Example of Length Determination

A sensing device composed of two microfluidic chambers, one or morefluidic channels connecting the two microfluidic chambers, and a pair ofsensing electrodes disposed along the length of each fluidic channel, isfilled with an ionic fluid. Typically, the fluid may be water thatcontains salt.

Multiple copies of a fragment of DNA of unknown length may be introducedinto one of the microfluidic chambers that is connected to the fluidicchannel that contains a pair of sensing electrodes. Macroscopicelectrodes are used to electrophorese the DNA strands from themicrofluidic chamber into one or more fluidic channels. As the DNAenters the fluidic channel, it assumes a linear conformation. The degreeto which it is linearized depends on a number of factors. Some of thosefactors are, e.g., the persistence length of the DNA strand, thetemperature, the ionic conditions, and the width and depth of thefluidic channel.

The potential applied by the electromotive electrodes causes the DNAstrand to move down the length of the fluidic channel. As the fragmentmoves down the fluidic channel it passes through the volume betweensensing electrodes. When the leading edge of the DNA enters a volumebetween sensing electrodes, a change in some electrical characteristicsuch as cross channel current or potential between two sensingelectrodes may be recorded. The recorded signal is composed of a timestamp and an indication of change in potential or other electricalproperty. The value of the electrical property may also be recorded. Thevalue may be subtracted from the background signal or may be an absolutevalue. A table may be generated by a computer that lists all responsesoccurring in the volume between sensing electrodes and the time stampfor each response. A computer program may subsequently determine theduration of the signal. As the trailing edge of the DNA strand exits thevolume between sensing electrodes, the electrical response typicallyreturns to the value which was observed before the DNA entered thevolume. The magnitude of the electrical response depends on theexperimental set-up; preferably, the electrical response is equal to atleast 3 times the magnitude of the root mean square noise for thesystem.

A calibrated standard curve may be applied to the measured length inorder to calculate the true length of the analyte. For example, thedevice may be calibrated with a series of DNA fragments of known lengththat are electrophoresed through the fluidic channel under the sameconditions, e.g., ionic strength, temperature, pH as the analyte. Thefragments preferably span enough different lengths to cover the rangethat may be used in the experiment to measure the length of the unknownfragment.

Example of DNA Sequencing

A target DNA strand of known or unknown sequence may be denatured.Denaturation of the duplex DNA is typically accomplished through theapplication of heat or chemicals, such that the hydrogen bonds betweenpaired strands are broken. The denatured DNA sample is incubated with aprobe of known sequence and base length or divided for incubation withmultiple probes, each with their own specific recognitions sequences onthe target DNA. In order to hybridize the probe or probes to theirrecognition sequence or sequences, the conditions for the incubation arechosen such that the probe or probes bind to the known specificrecognition site in preference to other sites or mismatch sites. Theconditions are also chosen so that more of the probe binding sites onthe denatured DNA strands are bound to a probe than unbound. Thesolution may be a buffered ionic solution. The solution may be agitatedto facilitate binding of the probes. The temperature of the solution maybe varied during the course of the incubation. For instance, thetemperature of the incubation may be slowly cooled over the course ofthe hybridization.

Once the denatured target DNA has been hybridized with a probe orprobes, the sample is introduced into a microfluidic chamber at one endof the fluidic channel device. The fluidic channel device is filled withan ionic solution, e.g., a salt solution. The solution may also bebuffered. The excess probe or probes may be removed prior to theintroduction of the sample into the microfluidic chamber. Gel filtrationis one method for removing short probes from a longer strand of DNA.Alternatively, other commercially available purification methods areavailable. Once the target DNA strand with hybridized probes has beenintroduced into a microfluidic chamber, a potential is applied viaelectromotive electrodes to drive the DNA from the microfluidic chamberinto one or more fluidic channels.

The target DNA, upon entering the fluidic channel, typically assumes alinearized conformation. The narrower the fluidic channel, the morelinearized the DNA is forced to become. The voltage applied to themacroscopic electromotive electrodes electrophoretically drives the DNAdown the fluidic channel. As the DNA and hybridized probes move down thefluidic channel they enter the volume between sensing electrodes in thefluidic channel.

In the absence of DNA, the volume between sensing electrodes may containonly the ionic solution and have a baseline potential differencemeasured between the two sensing electrodes. As DNA enters the volumebetween sensing electrodes, the potential measured between the twosensing electrodes changes because the DNA has a conductivity differentfrom that of the ionic solution. When DNA enters the volume betweensensing electrodes, the conductivity of the channel between the twosensing electrodes is typically reduced with respect to the conductivitywhen only ionic fluid is present between the sensing electrodes. When aportion of the DNA that also has a probe hybridized thereto enters thevolume between sensing electrodes, the potential changes further.

As the molecule passes between sensing electrodes, the monitored voltagevaries by a detectable and measurable amount. The electrodes detect andrecord this variation in voltage as a function of time. These variationsin voltage are the result of the relative diameter of the molecule thatis passing between sensing electrodes at any given time. For example,the portions of the biomolecule that have probes bound thereto are twicethe diameter of the portions of the biomolecule that have not beenhybridized and therefore lack probes.

This relative increase in volume of the biomolecule passing betweensensing electrodes causes a temporary increase in resistance betweensensing electrodes resulting in a measurable voltage variation. As theportions of the biomolecule that include probes pass between sensingelectrodes, the current is further impeded, forming a relative spike inthe recorded voltage during passage of the bound portion, whichdecreases again after the hybridized portion has passed. The sensingelectrodes detect and reflect these variations in the monitored current.Further, the measurements of the voltage variations are measured andrecorded as a function of time. As a result, the periodic interruptionsor variations in resistance indicate where, as a function of relative orabsolute position, the known probe sequence has attached to thebiomolecule.

When the DNA or a probe on the target DNA enters a fluidic channel, anelectrical signal is recorded. The electrical signal is composed of atime stamp and the value of the changed electrical property. Theelectrical property value may be subtracted from the background signalor may be an absolute value. A table may be generated by a computer thatlists all responses occurring between sensing electrodes and the timestamp for each response. A computer program may subsequently determinethe length of the biopolymer and the location of the hybridized probeson the biopolymer. The location of the probe on the biopolymer may bedetermined in terms of nanometers, base pairs or percentage of totalbiopolymer length.

The probe's location on the biopolymer can be determined according toits distance from the end of the biopolymer. This may be done through adetermination of a total length of the biopolymer using a calibratedstandard. The duration of the biopolymer signal may be compared to acalibrated standard curve in order to calculate the true length of theanalyte. For example, the device may be calibrated with a series of DNAfragments of known length that are electrophoresed through the fluidicchannel under the same conditions as the analyte, e.g., ionic strength,temperature, pH. The fragments preferably span enough different lengthsto calibrate the sensing electrodes to measure the length of the unknownfragment.

More of the sequence can be determined through subsequent or parallelhybridization with a second plurality of probes and the detecting,analyzing, and determining steps may be repeated with the subsequentplurality of probes. The designs described herein merge nanopore andfluidic channel technologies and decouple the driving electrophoreticforce from the detected signal. By using voltage sensing and byfabricating voltage amplifiers directly on the substrate where thesensing electrodes are placed, the device may operate at higherfrequencies than has been possible with previous geometries.

The described embodiments of the invention are intended to be merelyexemplary and numerous variations and modifications will be apparent tothose skilled in the art. All such variations and modifications areintended to be within the scope of the present invention as defined inthe appended claims.

1. A device for voltage sensing of analytes, the device comprising: afluidic channel defined in a substrate; a pair of sensing electrodesdisposed in the channel for sensing voltage therein, the pair of sensingelectrodes comprising a first and a second sensing electrode disposed attwo discrete locations along a length of the fluidic channel; and a pairof electromotive electrodes disposed at a first end and a second end ofthe fluidic channel for applying a potential along the fluidic channel,wherein the fluidic channel comprises a nanochannel or a microchannel.2. The device of claim 1, wherein the substrate comprises a materialselected from the group consisting of silicon, silicon dioxide, fusedsilica, and gallium arsenide.
 3. The device of claim 1, wherein each ofthe sensing and electromotive pairs of electrodes comprises a materialselected from the group consisting of platinum, gold, chrome, titanium,silver chloride, silver, and graphene.
 4. The device of claim 1, whereinthe first sensing electrode is disposed on a first side of the fluidicchannel and the second sensing electrode is disposed on an opposing sideof the fluidic channel.
 5. The device of claim 1, wherein each of thefirst and second sensing electrodes is disposed on a first side of thefluidic channel.
 6. The device of claim 1, wherein each of the first andsecond sensing electrodes transverses the fluidic channel.
 7. The deviceof claim 1, wherein the first sensing electrodes transverses the fluidicchannel and the second sensing electrode is disposed on a side of thefluidic channel.
 8. The device of claim 1, wherein the electromotivepair of electrodes comprises macroscopic electrodes for generating aconstant, changing, or oscillating electrophoretic force in the fluidicchannel for translocation of an analyte disposed therein.
 9. The deviceof claim 1, further comprising a measurement tool for measuring avoltage sensed by the pair of sensing electrodes.
 10. The device ofclaim 9 where the measurement tool comprises a voltmeter.
 11. The deviceof claim 1, further comprising a plurality of fluidic channels.
 12. Thedevice of claim 1, further comprising a voltage amplifier disposed onthe substrate.
 13. The device of claim 1, wherein the fluidic channelhas a width selected from a range of 1 nm to 5 μm.
 14. The device ofclaim 1, wherein the fluidic channel has a depth selected from a rangeof 1 nm to 5 μm.
 15. The device of claim 1, wherein the fluidic channelhas a length selected from a range of 1 μm to 10 cm.
 16. A method fordetecting an analyte, the method comprising the steps of: disposing theanalyte in a fluidic channel; applying a potential along the fluidicchannel; translocating the analyte from a first end of the fluidicchannel to a second end of the fluidic channel; and measuring a voltagesignal between a pair of sensing electrodes disposed in the fluidicchannel as the analyte moves past the pair of sensing electrodes, thepair of sensing electrodes comprising a first and a second electrodedisposed at two discrete locations along a length of the fluidicchannel, wherein the fluidic channel comprises a nanochannel or amicrochannel.
 17. The method of claim 16, wherein applying a potentialalong the fluidic channel comprises generating an electrophoretic forcetherein.
 18. The method of claim 16, wherein translocating the analytecomprises using a pressure differential.
 19. The method of claim 16,wherein translocating the analyte comprises using a chemical gradient.20. The method of claim 16, wherein the analyte comprises a biopolymerselected from the group consisting of deoxyribonucleic acids,ribonucleic acids, and polypeptides.
 21. The method of claim 20, whereinthe biopolymer comprises a single-stranded molecule.
 22. The method ofclaim 16, wherein the analyte comprises a biopolymer having at least oneprobe attached thereto.
 23. The method of claim 22, wherein the voltagesignal changes when the biopolymer moves through a volume between thesensing electrodes and further changes when the portion of thebiopolymer containing the probe moves through the volume between thesensing electrodes.
 24. The method of claim 23, further comprisingrecording a time between voltage signal changes.
 25. The method of claim24, wherein a duration of a change in the voltage signal indicates apresence of a probe, further comprising using the voltage signal todetermine a distance between two probes on the biopolymer.
 26. Themethod of claim 16, further comprising using a duration of a change inthe voltage signal to determine a length of the analyte.
 27. A methodfor determining a sequence of a biopolymer, the method comprising:preparing an analyte comprising the biopolymer; disposing the analyte ina fluidic channel; applying a potential along the fluidic channel;translocating the analyte from a first end of the fluidic channel to asecond end of the fluidic channel; and measuring a voltage signalbetween a pair of sensing electrodes disposed in the fluidic channel asthe analyte moves past the pair of sensing electrodes, the voltagesignal corresponding to locations along the biopolymer, the pair ofsensing electrodes comprising a first and a second electrode disposed attwo discrete locations along a length of the fluidic channel, whereinthe fluidic channel comprises a nanochannel or a microchannel.
 28. Themethod of claim 27, wherein applying a potential along the fluidicchannel comprises generating an electrophoretic force therein.
 29. Themethod of claim 27, wherein translocating the analyte comprises using apressure differential.
 30. The method of claim 27, wherein translocatingthe analyte comprises using a chemical gradient.
 31. The method of claim27, wherein preparing the analyte comprises hybridizing the biopolymerwith a probe.
 32. The method of claim 31, wherein a change in thevoltage signal corresponds to a location along the hybridized biopolymercontaining the probe.
 33. The method of claim 27, further comprisingprocessing the voltage signal using a computer algorithm to reconstructthe sequence of the biopolymer.
 34. The method of claim 27, wherein thebiopolymer comprises a double-stranded biopolymer target molecule. 35.The method of claim 34, wherein preparing the analyte comprisescontacting the target molecule with a first probe having a first probespecificity for recognition sites of the target molecule to form a firstplurality of local ternary complexes, the first probe having a firstpredicted recognition site sequence.
 36. The method of claim 35, furthercomprising using the voltage signal to determine positional informationof the first plurality of local ternary complexes.
 37. The method ofclaim 36, wherein the positional information includes a parametercorresponding to a spatial distance between two local ternary complexes.38. The method of claim 36, wherein preparing the analyte furthercomprises contacting the target molecule with a second probe having asecond probe specificity for recognition sites of the target molecule toform a second plurality of local ternary complexes, the second probehaving a second predicted recognition site sequence.
 39. The method ofclaim 38, further comprising using the voltage signal to determinepositional information of the second plurality of local ternarycomplexes.
 40. The method of claim 39, further comprising aligningpositional information of at least the first and second plurality oflocal ternary complexes to determine a DNA sequence of the target. 41.The method of claim 27, wherein the biopolymer comprises adouble-stranded nucleic acid target molecule having a plurality ofbinding sites disposed along the sequence thereof.
 42. The method ofclaim 41, wherein preparing the analyte comprises adding a plurality ofprobe molecules having a first sequence specificity to thedouble-stranded nucleic acid target molecule.
 43. The method of claim42, further comprising incubating the probe molecules having the firstsequence specificity and the target molecule so as to effectuatepreferential binding of the first probe molecules to both a firstbinding site and a second binding site of the target molecule.
 44. Themethod of claim 43, further comprising using the voltage signal tomeasure a parameter related to a distance between the first binding siteand the second binding site.
 45. The method of claim 27, whereinpreparing the analyte comprises contacting the biopolymer with a firstprobe to create at least one probe-target complex at a recognition siteof the biopolymer for which the first probe has a known specificity,while leaving uncomplexed, regions of the biopolymer for which the firstprobe is not specific.
 46. The method of claim 45, wherein preparing theanalyte further comprises contacting the biopolymer with a second probeto create at least one probe-target complex at a recognition site of thebiopolymer for which the second probe has a known specificity, whileleaving uncomplexed, regions of the target for which the second probe isnot specific.
 47. The method of claim 46, further comprising using thevoltage signal to detect and record complexed and uncomplexed regions ofthe biopolymer to create a first probe map of the first probe and asecond probe map of the second probe, the first probe map and the secondprobe map incorporating information on the relative position of thehybridization of the probes.
 48. The method of claim 47, furthercomprising determining a candidate sequence by ordering at least twoprobe sequences using positional information or a combination ofoverlapping sequences of the probe molecules and positional information.49. The method of claim 46, wherein the first and second probe mapsfurther incorporate information on an error of the positionalinformation for each probe.
 50. The method of claim 49, furthercomprising determining a candidate sequence by ordering at least twoprobe sequences using positional information and parameters relating tothe error in positional information or a combination of overlappingsequences of the probe molecules and positional information and error inpositional information.